Formation of membrane domains during the activation of protein kinase C
Li, Yang
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Permalink
https://hdl.handle.net/2142/20918
Description
Title
Formation of membrane domains during the activation of protein kinase C
Author(s)
Li, Yang
Issue Date
1995
Doctoral Committee Chair(s)
Glaser, Michael
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Language
eng
Abstract
The relationship between lipid domains and protein kinase C activity was studied via the direct visualization and quantitation of domains by fluorescence digital imaging microscopy. The lateral organization of protein kinase C, its substrate (the MARCKS peptide) and its three activators (phosphatidylserine, Ca$\sp{2+}$ and DAG) in large unilamellar vesicles were investigated. The formation of phosphatidylserine domains could be induced by either Ca$\sp{2+},$ the MARCKS peptide, or protein kinase C. However, only Ca$\sp{2+}$ could induce diacylglycerol to partition into the phosphatidylserine domains. In the complete protein kinase C assay mixture, two separate triple-labeling experiments demonstrated the colocalization of phosphatidylserine, protein kinase C, diacylglycerol, and the MARCKS peptide in domains. The amounts of all the labeled components in whole vesicles and in domains were measured at various concentrations of either phosphatidylserine, Ca$\sp{2+},$ diacylglycerol, the MARCKS peptide or with the addition of poly-lysine. The role of each component in forming membrane domains and in mediating the enzyme activity was analyzed. The results indicated that the inclusion of the MARCKS peptide in the domains, not just the binding of the substrate to vesicles, was especially important for PKC activity. Increasing the ionic strength disrupted the domains of the MARCKS peptide and phosphatidylserine and this was accompanied by a decrease in protein kinase C activity. Dansyl-poly-lysine, which inhibits protein kinase C, was similar to the MARCKS peptide in forming domains enriched in phosphatidylserine. The degree of enrichment of the MARCKS peptide in the phosphatidylserine domains decreased proportionally with protein kinase C activity when poly-lysine was added. Poly-lysine caused the MARCKS peptide to be displaced from the domains into the non-domain areas of the vesicles. Gramicidin, which was chosen to represent a neutral membrane protein, was excluded from the domains with phosphatidylserine and it had no effect on the enrichment of the domains or the enzyme activity. The formation of PKC domains required the presence of DAG and Ca$\sp{2+}$ in the physiological ionic strength. The PKC activity was proportional to the amounts of PKC and substrate in the domains. The results also showed that the MARCKS peptide left the domains after being phosphorylated. A model for the activation of protein kinase C involving sequestering of the reaction components into membrane domains is proposed.
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